CN113207912A - Chicken escherichia coli biological disinfectant and preparation and application thereof - Google Patents
Chicken escherichia coli biological disinfectant and preparation and application thereof Download PDFInfo
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- CN113207912A CN113207912A CN202110357130.0A CN202110357130A CN113207912A CN 113207912 A CN113207912 A CN 113207912A CN 202110357130 A CN202110357130 A CN 202110357130A CN 113207912 A CN113207912 A CN 113207912A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/40—Viruses, e.g. bacteriophages
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Abstract
The invention specifically provides a chicken escherichia coli biological disinfectant, which adopts three strains of chicken escherichia coli phages Ecp2, Ecp3 and Ecp6 as raw materials, and has a good disinfection effect on chicken barns with outbreak of chicken colibacillosis.
Description
Technical Field
The invention belongs to the technical field of biological engineering, relates to the technical field of microbial preparation, and particularly relates to a chicken escherichia coli biological disinfectant, and the technical field of preparation and application thereof.
Background
China is the first kingdom in the world, the total annual chicken raising amount exceeds 40 hundred million, the stock number of Xinjiang broilers is 4000-5000 ten thousand, and the stock number of laying hens is 700 plus 800 ten thousand. The chicken colibacillosis accounts for about 33 percent of the chicken bacterial diseases. 3107 thousands of chicken died due to colibacillosis every year in China, and the economic loss is more than 3 hundred million. According to incomplete statistics, the direct or indirect economic loss of the chicken colibacillosis to chicken farms accounts for 8-11% of the feeding cost. The traditional drug control method has the challenges of drug resistance, increased drug cost, environmental pollution and the like. In addition, the product has drug residue due to long-term use of antibacterial drugs, so that the quality is reduced, and the requirement of green and healthy culture is difficult to meet.
An important problem in the large-scale cultivation of livestock and poultry is the pollution problem in the cultivation process, which is not only a main source of national and rural environmental pollution, but also causes huge economic loss to farmers due to epidemic diseases caused by the cultivation environmental problem, and also has serious influence on the social economy, public safety and international trade of animals and animal products in China. A large amount of various chemical disinfectants are frequently used by a farm in order to control the environmental pollution problem, but the chemical disinfectants not only influence the animal body to cause production accidents such as disability and death of livestock and poultry, but also have adverse effects on the body health of farm workers to cause symptoms such as allergy and the like, and even more particularly can cause poisoning symptoms. In view of the problems caused by chemical disinfectants, a novel pollution-free disinfectant is urgently needed to replace the traditional disinfectant.
The bacteriophage has attracted great attention as a natural and safe antibiotic substitute for preventing and treating bacterial diseases, and the invention patent with the application number of 201210570103.2 provides a bacteriophage with environment disinfection capacity and application thereof, which has better lysis effect on enterobacteriaceae bacteria in a laboratory, but has larger difference of effect in practical use; the patent application No. 202010160390.4 discloses a coliphage composition and its application, the highest colibacillus bactericidal rate of the composition PD06+ PD114 is only 90.84%.
Based on the problem that the disinfectant condition of the phage biological agent to chicken colibacillosis infected chicken houses is not ideal, 7 strains of chicken pathogenic colibacillosis lytic phage are obtained by separating through a double-layer agar plate method in the early stage of the subject group and are named as Ecp1, Ecp2, Ecp3, Ecp4, Ecp5, Ecp6 and Ecp7 respectively, and the feeding effect is better by simply mixing drinking water for feeding. On the basis, in order to further improve the application of the phage biological preparation in broiler chicken breeding and highlight the economic added value, the phage biological disinfectant with better effect is searched.
Disclosure of Invention
Aiming at the technical problems that the existing phage preparation is not ideal in chicken colibacillosis infected chicken coop disinfection condition and long in disinfection action time in the prior art, the invention utilizes pathogenic colibacillosis lytic phage to carry out compatibility to prepare the chicken colibacillosis biological disinfectant. The invention aims to provide a chicken colibacillosis biological disinfectant, which has a good disinfection effect on chicken houses with outbreak of chicken colibacillosis, has a disinfection effect superior to that of ammonium bromide sunflower, is short in action time and quick in effect taking, can obviously reduce the total number of bacteria in the chicken houses, and has good effects on developing green and pollution-free environmental disinfectants and improving the economic benefits of broiler breeding.
The invention is realized by the following technical scheme:
the invention specifically provides a chicken escherichia coli biological disinfectant, which comprises escherichia coli phages Ecp2, Ecp3 and Ecp 6.
Preferably, the mixing volume ratio of the Escherichia coli bacteriophages Ecp2, Ecp3 and Ecp6 is 1:1 (3-1).
More preferably, the mixing volume ratio of the Escherichia coli phages Ecp2, Ecp3 and Ecp6 is 1:1: 1.
Preferably, the biological disinfectant for chicken escherichia coli has no phage titerLess than 108pfu/ml。
In the invention, three strains of Ecp2, Ecp3 and Ecp6 are adopted, the formed plaques and the specific forms are observed through purification and culture, and the formed plaques and the specific forms are identified as conventional Escherichia coli phages in combination with molecular level, the phages are all harmless and safe strains allowed to be used by the Ministry of agriculture, and can be obtained through public channels by ordinary technicians, and the culture conditions and the culture medium of the three strains of phages can be obtained by common reports in the field.
The invention provides application of the chicken escherichia coli biological disinfectant obtained by the preparation process in disinfection of chicken colibacillosis infected chicken houses.
The application of the chicken escherichia coli biological disinfectant in disinfection of chicken colibacillosis infected henhouses is spraying, and the dilution ratio is 1: 50.
The application of the chicken escherichia coli biological disinfectant in disinfection of chicken colibacillosis infected henhouses is that the spraying amount is 15ml/m3Once in the morning and evening.
By implementing the specific invention content of the invention, the following beneficial effects can be achieved:
(1) the invention aims to provide a chicken escherichia coli biological disinfectant, which has a good disinfection effect on chicken coops with outbreak of chicken colibacillosis, has a disinfection effect superior to that of ammonium bromide pelargonium bromide, is short in action time and quick in effect, can obviously reduce the total number of bacteria in the chicken coops, and has good effects on developing a green and pollution-free environment disinfectant and improving the economic benefits of broiler breeding.
(2) After the chicken colibacillus biological disinfectant is sprayed and disinfected for 30min, the bacterial quantity is reduced to 0.30 percent of that of a control group, and the bacterial quantity is only 0.10 percent of that of the control group after the chicken colibacillus biological disinfectant is sprayed for 70 min.
(2) Compared with the control group, the disinfection effect of the colibacillus provided by the invention on the ground of the chicken coop is remarkably different (P <0.05), and the disinfection effect of the phage group is remarkably different (P <0.001), which means that the phage serving as the disinfectant has more remarkable killing effect on the total number of bacteria than the colibacillus disinfectant.
(3) The chicken colibacillus provided by the invention has the following effects of sterilizing the air in the chicken coop: the number of the bacteria in the enterobacteriaceae family after the phage group is sterilized is very significantly lower than that in the control group (P <0.01), which indicates that the phage has very obvious sterilizing effect on the bacteria in the enterobacteriaceae family in the air sterilization of the henhouse.
(4) The chicken colibacillus provided by the invention has the following effects of sterilizing dust in chicken houses: the phage group has extremely obvious disinfection effect on the total number of bacteria in the henhouse dust and the enterobacteriaceae bacteria and is extremely superior to the sunflower methyl bromide disinfectant.
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Detailed Description
The present invention will be described below by way of examples, but the present invention is not limited to the following examples.
The reagents used in the present invention are: LB culture medium, sodium alginate, chitosan, calcium chloride and sodium citrate.
The equipment adopted in the invention comprises: single double-sided clean bench: shanghai Boxun industries, Inc. medical facilities; heir 4 ℃ refrigerator: shandong Qingdao Haier group; electric heating constant temperature water bath: the medical instrument factory of Yongguang, Beijing; constant temperature culture shake box: shanghai Zhicheng analytical instruments manufacturing, Inc.; a desk centrifuge: hettich, Germany; an ultrafreeze centrifuge: thermo in the united states; KDM type temperature adjustable electric heating jacket: south china heating equipment, jiangsu; an applicator; a constant-temperature incubator: shanghai sperm macro test Equipment, Inc.; electronic balance (JY1002 Shanghai precision scientific instruments Co., Ltd.); microfiltration membrane (0.22 μm): shanghai Xinya purification material factory (and the filter membrane is used after being installed and subjected to high pressure); a micro-pipette: U.S. eppendorf; a culture dish; a pressure cooker; vernier calipers, etc.
The reagents and materials can be purchased through public channels, and the equipment and instruments adopted in the process are all common equipment in the field.
All materials, reagents and equipment selected for use in the present invention are well known in the art, but do not limit the practice of the invention, and other reagents and equipment well known in the art may be suitable for use in the practice of the following embodiments of the invention.
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
The first embodiment is as follows: chicken escherichia coli biological disinfectant
The invention specifically provides a chicken escherichia coli biological disinfectant, which comprises escherichia coli phages Ecp2, Ecp3 and Ecp 6.
The mixing volume ratio of the Escherichia coli phage Ecp2, Ecp3 and Ecp6 is 1:1 (3-1).
Example two: preparation method of chicken escherichia coli biological disinfectant
The invention specifically provides a preparation method of a chicken escherichia coli biological disinfectant, which comprises the following specific production steps:
taking 3 strains of bacteriophage which are separated to obtain Ecp2, Ecp3 and Ecp6 out of a refrigerator at the temperature of minus 80 ℃, amplifying and propagating the corresponding host bacteria by liquid LB for 8 to 12 hours, adding 1ml of each bacterium liquid and bacteriophage into a 5ml test tube filled with LB liquid culture medium, performing shake culture at the temperature of 37 ℃ and 160r/min for 8 to 12 hours, and determining the titer of the bacteriophage to reach 108And (4) pfu/ml or more, finishing the multiplication of the phage, and mixing according to the volume ratio to obtain the chicken colibacillus biological disinfectant.
Example three: chicken escherichia coli biological disinfectant
Taking 3 strains of bacteriophage which are separated to obtain Ecp2, Ecp3 and Ecp6 out of a refrigerator at the temperature of minus 80 ℃, amplifying and propagating the corresponding host bacteria by liquid LB for 8 to 12 hours, adding 1ml of each bacterium liquid and bacteriophage into a 5ml test tube filled with LB liquid culture medium, performing shake culture at the temperature of 37 ℃ and 160r/min for 8 to 12 hours, and determining the titer of the bacteriophage to reach 108And (4) pfu/ml or more, finishing the multiplication of the phage, and mixing according to the volume ratio of 1:1:3 to obtain the chicken colibacillus biological disinfectant.
Example four: chicken escherichia coli biological disinfectant
Taking 3 strains of bacteriophage which are separated to obtain Ecp2, Ecp3 and Ecp6 out of a refrigerator at the temperature of minus 80 ℃, amplifying and propagating the corresponding host bacteria by liquid LB for 8 to 12 hours, adding 1ml of each bacterium liquid and bacteriophage into a 5ml test tube filled with LB liquid culture medium, performing shake culture at the temperature of 37 ℃ and 160r/min for 8 to 12 hours, and determining the titer of the bacteriophage to reach 108And (4) pfu/ml or more, finishing the multiplication of the phage, and mixing according to the volume ratio of 1:1:2 to obtain the chicken colibacillus biological disinfectant.
Example five: chicken escherichia coli biological disinfectant
Taking 3 strains of bacteriophage which are separated to obtain Ecp2, Ecp3 and Ecp6 out of a refrigerator at the temperature of minus 80 ℃, amplifying and propagating the corresponding host bacteria by liquid LB for 8 to 12 hours, adding 1ml of each bacterium liquid and bacteriophage into a 5ml test tube filled with LB liquid culture medium, performing shake culture at the temperature of 37 ℃ and 160r/min for 8 to 12 hours, and determining the titer of the bacteriophage to reach 108And (4) pfu/ml or more, finishing the multiplication of the phage, and mixing according to the volume ratio of 1:1:1 to obtain the chicken colibacillus biological disinfectant.
Example six: determination of effect of mixing ratio of different phages
The chicken colibacillus biological disinfectant prepared in the third to fifth embodiments is selected for sterilization effect determination.
Adding 1ml of 45 chicken pathogenic escherichia coli suspension obtained by collecting and purifying collected chicken manure samples into a conical flask, and carrying out shake culture at 37 ℃ for 24 h. Taking 3 high-pressure EP tubes, adding 0.2ml of chicken colibacillus biological disinfectant prepared by mixing phage according to different proportions into each tube by using a micropipettor, fully mixing the mixture by slight oscillation, and standing for 10-15min at room temperature. Then the mixture of the host bacteria and the phage in the EP tube is subjected to inverted culture for 8-12h in a constant temperature incubator at 37 ℃, and then the clarity condition of the liquid is observed.
According to observation, the mixed phage liquid mixed according to the volume ratio of 1:1:3, 1:1:2 and 1:1:1 has a cracking effect on Escherichia coli purified in chicken manure samples, and has the characteristic of maintaining liquid clarification, wherein the liquid clarification can be maintained optimally by the mixed phage liquid mixed according to the volume ratio of 1:1: 1.
Example seven: determination of chicken colibacillus biological disinfectant disinfection effect
Based on the sixth embodiment, the biological disinfectant for chicken colibacillus prepared by the technical scheme in the fifth embodiment is selected for testing the disinfection effect.
Selecting 1 chicken coop with the outbreak of the chicken colibacillosis, and dividing the chicken coop into three regions which are not communicated with each other:
diluting the prepared chicken colibacillus biological disinfectant by sterile water, wherein the dilution ratio is 1:50, the spraying amount is 15ml/m3, and the prepared chicken colibacillus biological disinfectant is used for disinfecting the ground of a henhouse, the air of the henhouse and the dust of the henhouse once in the morning and at night to form a phage group;
adopting a conventional pelargonium bromide disinfectant disinfection method to disinfect the henhouse ground, the henhouse air and the henhouse dust to obtain a pelargonium bromide disinfectant group;
no treatment was done as a control group;
the total number of bacteria in the corresponding area of the colony house was determined 30min after sterilization and the differences between the treatment groups were compared. The results are as follows:
(1) spray disinfection effect of phage on host bacteria: after the phage is sprayed and disinfected for 30min, the bacterial quantity is reduced to 0.30% of that of a control group, the bacterial content shows a gradual descending trend along with the increase of action time, the bacterial quantity is only 0.10% of that of the control group after spraying for 70min, the spraying form of the phage has extremely obvious killing effect on host bacteria, and more than 99% of bacteria can be killed after 30 min.
(2) The effect of disinfecting the henhouse ground is as follows: compared with the control group, the disinfection effect of the ammonium bromide sunflower disinfectant group is remarkably different (P <0.05), and the disinfection effect of the phage group is remarkably different (P <0.001), which shows that the phage is used as the disinfectant and has more remarkable killing effect on the total number of bacteria than the ammonium bromide sunflower disinfectant.
(3) The chicken coop air disinfection effect is as follows: the number of the bacteria in the enterobacteriaceae family after the phage group is sterilized is very significantly lower than that in the control group (P <0.01), which indicates that the phage has very obvious sterilizing effect on the bacteria in the enterobacteriaceae family in the air sterilization of the henhouse.
The effect of disinfecting the dust in the henhouse is as follows: the phage group has extremely obvious disinfection effect on the total number of bacteria in the henhouse dust and the enterobacteriaceae bacteria and is extremely superior to the sunflower methyl bromide disinfectant.
As described above, the present invention can be preferably implemented, and the above-mentioned embodiments only describe the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various changes and modifications of the technical solution of the present invention made by those skilled in the art without departing from the design spirit of the present invention shall fall within the protection scope defined by the present invention.
Claims (7)
1. The chicken escherichia coli biological disinfectant is characterized by comprising the following components: comprises Escherichia coli phages Ecp2, Ecp3 and Ecp 6.
2. The biological disinfectant for chicken escherichia coli as recited in claim 1, wherein the mixed volume ratio of escherichia coli bacteriophage Ecp2, Ecp3 and Ecp6 is 1:1 (1-3).
3. The biological disinfectant for chicken escherichia coli as set forth in claim 1, wherein the mixed volume ratio of escherichia coli bacteriophage Ecp2, Ecp3 and Ecp6 is 1:1: 1.
4. The biological disinfectant for chicken escherichia coli as set forth in claim 1, wherein the phage titer in the biological disinfectant for chicken escherichia coli is not less than 108pfu/ml。
5. The use of a biological disinfectant of chicken escherichia coli prepared according to any one of claims 1 to 4 for disinfecting chicken barnacles infected chicken coops.
6. The application of the biological disinfectant for chicken escherichia coli prepared and obtained by the method in disinfection of chicken colibacillosis infected chicken houses, which is characterized in that the application mode is spraying, and the dilution ratio is 1: 50.
7. The application of the biological disinfectant for chicken escherichia coli prepared by the method in the disinfection of chicken barnacles infected chicken coops, which is prepared by the method of claim 5, wherein the spraying amount is 15ml/m3Once in the morning and evening.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113230283A (en) * | 2021-04-01 | 2021-08-10 | 石河子大学 | Chicken escherichia coli therapeutic microecological preparation and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103289963A (en) * | 2012-12-12 | 2013-09-11 | 青岛农业大学 | Bacteriophage with environment disinfection capability and applications thereof |
| WO2016036503A1 (en) * | 2014-09-04 | 2016-03-10 | Arizona Board Of Regents On Behalf Of Arizona State University | Antigen compositions and methods of use for treating extraintestinal pathogenic e. coli infections |
| CN111226986A (en) * | 2020-01-20 | 2020-06-05 | 上海交通大学 | Spray disinfectant containing culture environment phage composition, preparation method and application thereof |
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- 2021-04-01 CN CN202110357130.0A patent/CN113207912A/en active Pending
Patent Citations (3)
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|---|---|---|---|---|
| CN103289963A (en) * | 2012-12-12 | 2013-09-11 | 青岛农业大学 | Bacteriophage with environment disinfection capability and applications thereof |
| WO2016036503A1 (en) * | 2014-09-04 | 2016-03-10 | Arizona Board Of Regents On Behalf Of Arizona State University | Antigen compositions and methods of use for treating extraintestinal pathogenic e. coli infections |
| CN111226986A (en) * | 2020-01-20 | 2020-06-05 | 上海交通大学 | Spray disinfectant containing culture environment phage composition, preparation method and application thereof |
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| 刘功成: "裂解性噬菌体与抗菌肽在肉鸡养殖中的应用分析", 《中国优秀博硕士学位论文全文数据库(硕士) 农业科技辑》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113230283A (en) * | 2021-04-01 | 2021-08-10 | 石河子大学 | Chicken escherichia coli therapeutic microecological preparation and application thereof |
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Application publication date: 20210806 |